SUMMARY DNA double-strand break (DSB) repair is essential to prevent persistent DNA damage, counteract genome instability and suppress tumor development. The spatial organization of DNA repair is critical to protect the genome from chromosome translocations and complex rearrangements. Thus, it is critical to better understand the mechanisms underlying the formation of DNA repair domains and the contribution of these domains to the maintenance of genome stability and, ultimately, tumor suppression. Armed with novel tools to manipulate the formation of homology-directed repair domains, we propose three specific aims to assess the impact of perturbing their formation on DNA repair and genome stability: mutation generation and chromosome rearrangements. First, we will investigate further how actin movements generated by the WASP-ARP2/3 pathway participate in DSB repair domain formation using ARP2/3 inhibition or clinically relevant mutations in WASP. Repair domains will be probed by state-of-the-art microscopy as well as by chromosome conformation capture assays (Hi-C). Next, we will assess the consequences of interfering with DNA repair domain formation on genome stability, specifically on chromosome translocations arising from various genomic insults: endonucleases, topoisomerase inhibitors and replication stressors. Finally, we will define the mutational signatures associated with DSB repair domain defects using loss and gain of function mutations in the WASP-ARP2/3 pathway. This project will greatly benefit from conceptual and technical interactions with all other projects of the program. The proposed experiments should provide an unprecedented level of understanding of the impact of the spatial organization of the nucleus, i.e. repair domains, on genome stability and subsequent tumor development.